Endoreduplication is a type of cell cycle in which nuclear chromosome DNA duplication takes place without cell division. When endoreduplication is repeated, the nuclear DNA content (i.e., a nuclear phase) is doubled from the basic 2C, and cells having doubled 4C or 8C nuclear DNA content are produced. Cells are known to grow in response to an increase in the nuclear DNA content. Since the size of an organism is determined based on the number and the sizes of cells constituting an individual, endoreduplication is considered to be a mechanism that determines organism size.
Although endoreduplication is observed in several tissues in insects and mammals, this feature is a characteristic of plant organs, and it can serve to distinguish plant development from that of other organisms. In plants, many organs are composed of a mixture of cells of different ploidy levels, and this feature is prominent in hypocotyl elongation, leaf expansion, and endosperm development. These polyploid cells are commonly observed in various multicellular organisms, such as insects, mammals, and plants (Non-Patent Documents 1 and 2). Polyploid cells are often seen in various developing tissues and are correlated with development; hence, polyploidy is thought to be a marker of differentiation (Non-Patent Document 3).
Hypocotyl elongation of seedlings is a typical size increment caused by endoreduplication in Arabidopsis thaliana. Cells contain as much as 8C (C is a set of haploid chromosomes) of nuclear DNA in light-grown seedlings and as high as 16C in dark-grown seedlings (Non-Patent Document 4). The polyploidy levels in hypocotyls are also known to be controlled by phytohormones (Non-Patent Document 5). Constitutively triple response 1 (ctr1) is an ethylene signal transduction mutant in which the ethylene signal is constitutively activated and causes a triple response without exogenous ethylene (Kieber, J. J., Rothenberg, M., Roman, G, Feldmann, K. A., and Ecker, J. R., 1993, CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the raf family of protein kinases, Cell 72, 427-441), and ctr1 has increased polyploidy levels, as high as 32C, in hypocotyls of dark-grown seedlings (Non-Patent Document 6). This indicates that ethylene regulates endoreduplication positively in hypocotyl cells.
Endoreduplication is also involved in the development of a plant's organs. A trichome consists of a single cell that contains a nucleus of up to 32C (Non-Patent Document 7). Endoreduplication is also observed in endosperm, and there are several reports of the involvement of cell-cycle-related genes in endosperm expansion (Non-Patent Documents 8 and 9).
Thus, the regulation of endoreduplication plays an important role in plant development and differentiation.
To date, cell-cycle-related factors are known to control endoreduplication, and a representative example is cyclin. For example, the D-type cyclin gene CYCD3;1 expresses specifically in meristems and developing leaves in Arabidopsis. When CYCD3;1 is overexpressed, the polyploidy levels of transgenic plants are reduced and cell sizes become smaller (Non-Patent Document 10). This indicates that CYCD3;1 is involved in cell proliferation through inhibiting endoreduplication in plant tissue. Also, it is reported that an Arabidopsis thaliana A-type cyclin gene, CYCA2;1, is expressed in various cells, such as guard cells, where substantially no endoreduplication occurs (Non-Patent Documents 11 and 12). When tobacco (Nicotiana tabacum) CYCA3;2, which is also an A-type cyclin gene, is overexpressed in Arabidopsis, polyploidy levels are reduced in various tissues (Non-Patent Document 13). It is also reported that loss of Arabidopsis thaliana CYCA2;3 function increases polyploidy in mature true leaves (Non-Patent Document 14). In particular, accordingly, A-type cyclins can play an important role in regulating endoreduplication in plants.
Although there have been several research reports regarding endoreduplication as described above, the major part of the mechanism of endoreduplication in plants has not yet been elucidated. Thus, elucidation of such mechanism enables understanding of the mechanism of plant size determination, which in turn realizes various applications.                (Non-Patent Document 1) Edgar, B. A., and On-Weaver, T. L. (2001) Endoreplication cell cycles: more for less. Cell 105, 297-306.        (Non-Patent Document 2) Joubes, J., and Chevalier, C. (2000) Endoreduplication in higher plants. Plant Mol. Biol. 43, 735-745.        (Non-Patent Document 3) De Veylder, L., Beeckman, T., Beemster, G. T., Krols, L., Terras, F., Landrieu, I., van der Schueren, E., Maes, S., Naudts, M., and Inzé, D. (2001) Functional analysis of cyclin-dependent kinase inhibitors of Arabidopsis. Plant Cell 13, 1653-1668.        (Non-Patent Document 4) Gendreau, E., Traas, J., Desnos, T., Grandjean, O., Caboche, M., and Hofte, H. (1997) Cellular basis of hypocotyl growth in Arabidopsis thaliana. Plant Physiol. 114, 295-305.        (Non-Patent Document 5) Gendreau, E., Orbovic, V., Hofte, H., and Traas, J. (1999) Gibberellin and ethylene control endoreduplication levels in the Arabidopsis thaliana hypocotyl. Planta 209, 513-516.        (Non-Patent Document 6) Gendreau, E., Traas, J., Desnos, T., Grandjean, O., Caboche, M., and Hofte, H. (1997) Cellular basis of hypocotyl growth in Arabidopsis thaliana. Plant Physiol. 114, 295-305.        (Non-Patent Document 7) Melaragno, J. E., Mehrotra, B., and Coleman, A. W. (1993) Relationship between endopolyploidy and cell size in epidermal tissue of Arabidopsis. Plant Cell 5, 1661-1668        (Non-Patent Document 8) Sun, Y, Flannigan, B. A., and Setter, T. L. (1999) Regulation of endoreduplication in maize (Zea mays L.) endosperm. Isolation of a novel B1-type cyclin and its quantitative analysis. Plant Mol. Biol. 41, 245-258.        (Non-Patent Document 9) Larkins, B. A., Dukes, B. P., Dante, R. A., Coelho, C. M., Woo, Y. M., and Liu, Y. (2001) Investigating the hows and whys of DNA endoreduplication. J. Exp. Bot. 52, 183-192.        (Non-Patent Document 10) Dewitte, W., Riou-Khamlichi, C., Scofield, S., Healy, J. M., Jacqmard, A., Kilby, N. J., and Murray, J. A. H. (2003) Altered cell cycle distribution, hyperplasia, and inhibited differentiation in Arabidopsis caused by the D-type cyclin CYCD3. Plant Cell 15, 79-92        (Non-Patent Document 11) Melaragno, J. E., Mehrotra, B., and Coleman, A. W. (1993) Relationship between endopolyploidy and cell size in epidermal tissue of Arabidopsis. Plant Cell 5, 1661-1668.        (Non-Patent Document 12) Burssens, S., de Almeida Engler, J., Beeckman, T., Richard, C., Shaul, O., Ferreira, P., Van Montagu, M., and Inze, D. (2000) Developmental expression of the Arabidopsis thaliana CycA2;1 gene. Planta 211, 623-631.        (Non-Patent Document 13) Yu, Y, Steinmetz, S., Meyer, D., Brown, S., Shen, W. H. (2003). The tobacco A-type Cyclin, Nicta; CYCA3;2, at the nexus of cell division and differentiation. Plant Cell 15, 2763-2777.        (Non-Patent Document 14) Imai, K. K., Ohashi, Y, Tsuge, T., Yoshizumi, T., Matsui, M., Oka, A., and Aoyama, T. (2006) The A-Type Cyclin CYCA2;3 Is a Key Regulator of Ploidy Levels in Arabidopsis Endoreduplication. Plant Cell 18, 382-396.        